There is a fundamental gap in understanding how lateralized neural activity, and anatomical- and physiological neural plasticity affect language acquisition. The plasticity that is necessary to acquire a second language (L2) later in childhood is even less well understood. Thus, it is challenging to identify the cause of delays in L2 learning at the neural level. The acquisition of a songbird's song parallels human speech learning at the behavioral as well as the neural level and thus provides unique opportunities to investigate the neural mechanisms of learning and memory. The long-term goal is to determine the cellular and system-level mechanisms through which birds acquire, store, and retrieve auditory memories. The objective of the proposed renewal application is to determine the neural systems involved in vocal plasticity related to imitating elements from a second song model (S2) later in development. The central hypothesis, formulated on the basis of preliminary data, is that lateralized neural plasticity is necessary for successful acquisition of multiple auditory memories. This hypothesis will be tested by pursuing two specific aims, which will: 1) Define lateralization of brain activity in relation to vocal plasticity during sensorimotor learning; and 2) Determine the impact of physiological plasticity on vocal learning. In the first aim, manipulations of the early auditory environment will be combined with analysis of song learning and quantification of the Blood Oxygenation Level Dependent (BOLD) response. Strong preliminary data provide evidence that zebra finches can learn elements from different vocal models at two time points in development, and pilot fMRI studies indicate feasibility to perform the proposed studies in the applicant's laboratory. In the second aim, classification of inhibitory cell types with immunohistochemistry will be used at critical moments in development; song learning from two different vocal models will be quantified and the inhibitory cell types that are contributing to vocal plasticity will be determined. Each of these methods, including the use of commercially available antibodies and freely available song and image analysis software, has previously been established in songbirds, which, in combination with data from pilot studies, provides evidence for the feasibility of the proposed studies in the PI's lab. The approach is innovative, as by combining longitudinal functional imaging and triple labeling methods, it is overcoming inherent limitations to studying the fundamental processes underlying neural plasticity for L2 learning in humans by using an established animal model. The proposed research is significant, because by using approaches not available in humans, we will gain a mechanistic understanding of the plasticity that underlies formation of multiple auditory memories in vocal learners. This will provide an understanding of adaptive sensorimotor integration contributing to vocal behavior throughout an organism's lifetime.